RqN]
T--)
Hf
+
R+N]
N
I H
N
attempts were made to use a strong anionic resin to separate the imidazoline from its degradation product. A 10% (v/v) methanolic pH 12.4 buffer as the mobile phase was suitable for separation of xylometazoline from its degradation product (Figure 6) and quantitation of the active ingredient (Table VI). Although other imidazolines are not chromatographed in this fashion, this method can obviously be extended to them. It offers a ready alternative if one encounters interference from the excipients or ingredients in a formulation. Each method does provide the specificity and speed and can be considered a method of choice for these drugs. The proposed methods are satisfactory for separation of imidazolines and suffer only one disadvantage in that the resins used are not stable for extended periods as indicated in the manufacturer's product bulletins (21, 22) a t the
pH values employed, but their life was extended somewhat by using temperatures below 45 " C and by eluting with water immediately after use. Experience in our laboratory has shown that the life of the column depends not only on the pH of the mobile phase but the length of time the column is exposed to the buffer, the temperature of the column and mobile phase, and the frequency of washing with water. Columns have been used from several weeks to several months. The relatively short column life was not considered to be a disadvantage as the increased precision and accuracy and speed over existing methods more than compensated for this. Received for review March 5 , 1973. Accepted April 23, 1973. (21) "Strong Anion Exchange Column for High Speed Liquid Chromatography," Bulletin dated 10/1/69, DuPont Instrument Company, Wilmington, Del., 19898. (22) "Strong Cation Exchange Column for High Speed Liquid Chromatography," Product Report dated 3/30/70, DuPont Instrument Company, Wilmington,-Del., 19898.
Flow Coulometric Detector for Liquid Chromatography Yoshinori Takata Hitachi Research Laboratory, Hitachi Ltd., Hitachi, Ibaragi, Japan
Giichi Muto Institute of Industrial Science, University of Tokyo, Tokyo, Japan
A new method of detection based on constant potential coulometry for liquid chromatography has been developed. This paper presents an improvement of the electrolytic flow cell for the detection of sample constituents in the column effluent. The new cell had a response time of within 1 second and could be used in the flow rate of effluent as large as 6 ml/min with the electrolytic efficiency of more than 99.5%. Primary, secondary, and indirect coulometry were applied to the detection of the materials such as metal ions, inorganic anions, organic acids, phenols, and sugars. The method was applied to the detection of 5 X to 5 X 1 0 - l o mole of these substances separated by ion-exchange chromatography with good results.
A new type of cell which is considerably more efficient than the one reported earlier (5-7) is devised and tested on the detection of metal ions, halide ions, amino acids, carboxylic acids, phenols, and sugars. In principle, the technique depends on introducing a column effluent into a cell where electrochemical reactions of almost all of the objective components take place a t a working electrode of constant potential. The potential selected is of sufficient magnitude to effect the reaction of the objective but is kept at a lower magnitude than required for the reaction with the eluate substrate. The electrochemical reactions for the detection are as follows: kIg-DTPAl3Ag
+
+
Mn* + 2e AgX
X--
-
[CU(SCN)~I- + 2Am--
At present, several optical, thermal, and electrochemical detectors are available for liquid chromatography ( I 4 ) . However, these detectors present considerable technical difficulties to determine many metals or organic acids with high sensitivity, The present paper describes progress in applying the constant potential coulometric technique to the analysis of column effluents in liquid chromatography. (1) S. H . Byrne, Jr.. "Modern Practice of Liquld Chromatography." J. J. Kirkland, Ed., Wiley-lnterscience, New York, N.Y.. 1971, pp 95-
A N A L Y T I C A L C H E M I S T R Y , VOL. 45,
+
2H'
+
2e
-
H2Q
(1
1
(2) [CuAm21 PSCN-+ e
(3) (41 (5)
Equation 1 is utilized for the detection of metal ions, where DTPA means diethylenetriaminepentaacetate. Applied potential is considered to be a function of the con(5) Y. Takata and G .Muto, Jap. Anal., 14, 453 (1965) (6) Y. Takata and G. Muto. Jap. Anal., 1 5 , 269 (1966) (7) G . Muto and Y. Takata, "New Liquid Chromatography,'' Kagaku no Ryoiki. Extra No. 88, Nankodo, Tokyo, 1969, pp 188-201.
124.
(2) M. N. Munk. J. Chromatogr. Sci., 8, 491 (1970). (3) R. D. Conlon, Anal. Chem., 41 (4), 107A (1969). ( 4 ) J. F. K. Huber,J Chromatogr. Sci.. 7 , 172 (1969).
1864
Q
[M-DTPA](5-n)- + Hg
e
+
NO.
11, SEPTEMBER 1973
d
C
I
4 n
t
.
-e--I
w-
a--
g
b --+
El
Figure 1. Liquid
Electrode vessels
chromatograph with coulometric detector.
A : eluant, B : pulsation damper, C: column, D: coulometric detector cell, E , : electrolyte for working electrode reaction, €2: electrolyte for auxiliary electrode reaction, F : sample injector, G: source of electricity, H: recorder, P I : p u m p 1, PI:p u m p 2 (double channel peristaltic pump)
centration of metal ions (8). Equation 2 is for halide ions ( 9 ) , Equation 3 for amino acids, Am, and Equation 4 (10) for carboxylic acids, where Q and H2Q represent p-benzoquinone and hydroquinone, respectively. Further, for phenols, Equation 5 (11) is applied. The last equation is an example of indirect reaction which is for sugars. These reactions have been utilized for the detection and automatic recording of these objective species with high sensitivities.
EXPERIMENTAL Liquid Chromatograph. This work was performed with a Hitachi Liquid Chromatograph, Model 034, and a coulometric detector. A schematic diagram of the apparatus is shown in Figure 1. The eluant is led by means of pump 1 through the sampler to the water-jacketed glass column filled with ion-exchange resin, where objective components are separated. The effluent from the column, streaming in a Teflon tube of 0.65-mm i.d., is mixed with a suitable electrolyte before being passed through the detector cell, and the electrolytic current based on the objective electrochemical reaction is measured. The output of the electric circuit is recorded on a laboratory potentiometric recorder and the amounts of the components from the area of the peaks applying Faraday's law, are calculated. Detector Cell. Flow electrolytic cell, i.e., the detector cell, is illustrated in Figure 2. Shown in Figure 2 are vessels with an electrode made of carbon cloth (Tokai Electrode Mfg. Co., Ltd., Minato-ku, Tokyo; Type CH-n) or metal gauze tightly packed in a n opening of silicone rubber plate. The working electrode made of carbon cloth, platinum gauze, or silver wire netting is interposed between the auxiliary electrodes through diaphragms. The working electrode vessel is provided with a sample inlet and a n outlet. A sample to be analyzed is introduced into the working electrode vessel through the sample inlet and exhausted through the outlet. The auxiliary electrodes are silver-silver iodide wire nettings of about 40 mesh or carbon clothes. In order to make the electric resistance of the cell low, an ionexchange membrane is used as the diaphragm. The size of the openings is 48 mm in length, 15 mm in width, and 2 mm in thickness. These structual components are arranged and bound with clamping bolt. into a liquid-tight cell. (8) (9) (10) (11)
G . Muto and T. Kawaguchi,Jap. Anal., 17, 38 (1968). J. J. Linganeand L. A. Small, Anal. Chem., 21, 1119 (1949). J. C. Abbott and J. W. Collat, Anal. Chem.. 35, 859 (1963). K. Sasaki and W. J. N e w b y , J . Electroanal. Chem., 20, 137 (1969)
Figure 2. Flow
coulometric detector ce!l
a: Inlet of the cell, b : outlet of the celi, c, c ' : electrolyte inlet. d , d ' : electrolyte outlet, e: ion exchange membrane, f: auxiliary electrode, g: working electrode, h : case of the electrode vessels Electrical Circuit. In general, to effect constant potential coulometry, a potentiostat is necessary. However, the electrolytic cell prepared for liquid chromatography should be able to effect constant potential electrolysis by employing a source of substantially constant voltage of order of 0 to =t3 volts having a negligible impedance, because the fluid from the co!umn is not homogeneous and otherwise the working. electrode potential is not distributed uniformly. Electrolyte for Working Electrode Reaction. E1ectrolvf.e for the detection of metal io&: 0.01M Hg-DTPA-O.1M ammonium nitrate-1M ammonium hydroxide; for amino acids: 0.01M cuprous thiocyanate-4M potassium thiocyanate: for carboxylic acids: 0.01M p-benzoquinone-0.001M hydroquinone-0.1M potassium chloride; and for sugars: 0.1M potassium ferricyanide-3M sodium hydroxide were used. For halide ions and for phenols: no electrolyte was used. Electrolyte for Auxiliary Electrode Reaction. The electrolyte used for auxiliary electrode reaction was 0.5M potassium iodide when silver-silver iodide e!wtrode was used. Another electrolyte was 0.1M potassium ferricyanide-0.1M potassium ferrocyanide0.1M potassium nitrate. Column Resin. The strongly acidic cation exchange resin, Hitachi No. 2611 and No. 2613 having nominal 1 0 1 and 8% divinylbenzene cross linkage, and particle distributions of 15 f 2 p and 18 f 2 p , respectively, was obtained from Hitachi, Ltd. Aminex A-4 cation exchange resin 17 2 p was obtained from Bio-Rad Laboratories. These resins were purified by washing with 2M sodium hydroxide, water, 2M hydrochloric acid, and then with water again. The anion exchange resin were Bio-Rad AG 1-X4 and AG 1-X8 of -400 mesh. The resins were washed with 2M sodium hydroxide and with water. Chemicals. All chemicals were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan, and used as received.
*
RESULTS AND DISCUSSION Speed of Response. In regard to speed of response, the rapid speed makes the cell more widely applicable. Then, an impulse response characteristic was measured,-Le., an electrolyte was streamed into the cell and 5 ~1 of 0.02M Au(II1) ion was added into the conduit a t the point, 30 cm ahead of the inlet of the cell. In order to obtain the real response time of the cell, a value of the electrolysis time which is determined by extrapolating the How rate to infinity should be measured, because the peak obtained includes time loss by diffusion of the primary ion in the conduit.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
1865
20
10
0
Time ( m i n ) Figure 5. Separation and
detection of alkaline earth group
Sample size: 3 X l o - ' mole (Mg: 2.1 X l o - ' mole); resin: Aminex A-4; column: 54 mm X 6 mm-i.d., room temperature; eluant: 2M CH&OONH4,, pH 9.4; flow rate: 1 mi/min; electrolyte: 0.01M HgDTPA-1M NH4OH0.1M NH4N03; detection potential: 0.10 V vs. Ag-AgI
0
0
2
1
I/v Figure
3 4 (minim!)
3. Speed of response
Sample: 0.02M Au( I I I ) , 5 pI
0 Figure 6.
10
20 30 40 Time ( m i n 1
Heavy metal ions separation
Sample size: Hg 5.0, Cu 4 . 3 , Zn 4.7, Ni 6.4, Pb 7.1, Cd 4.7, and Co 4.1 X l o - ' mole; resin: Hitachi No. 2611 (degree of crosslinking: 10); column: 90 mm X 9 mm-i.d., room temperature: eluant: 0.15M Na-tartrate0.09M NaCI, pH 3.5; flow rate: 2 ml/min; electrolyte: 0.01M HgDTPA-1M NH40H-O.lM NH4N03; detection potential: 0.24 V vs. Ag-AgI
0 Figure 4.
1
2
3 4 5 6 Flow rate ( ml/ m i n )
Efficiency of the cell
Sample: 0.054 mg/ml Cu(ll)-0.25M CH3COONH4
In Figure 3, peak width a t half-height gained from the variation of the flow rate ploted against reciprocals of flow rate values of the eluate, is compared with the one of 2 mm in the light path of a photometrical cell of 60 pl inner volume. The graph shows that response time of the electrolytic cell is less than 1 second and is rapid enough as the detector for liquid chromatography. Efficiency of Electrolysis. If the response time is rapid enough and sufficient potential is applied to the working electrode, then the objective reaction will occur nearly perfectly. However, when the flow rate rises above such a speed that a part of the objective component flows through the cell without contacting the electrode, the efficiency of the electrolysis declines. 1866
Since accuracy of the quantitative analysis depends on the efficiency, and the amount of the substance is calculated from the peak area expressed with quantity of the electric charge by Faraday's law, assuming that 100% electrolysis is performed, it is important that it be electrolyzed completely. The relationship between flow rate of effluent and electrolytic efficiency was investigated for copper. The result is shown in Figure 4 comparing with the cell of a smaller working electrode. These values plotted were obtained from measuring the cupric ion poured out from the outlet of the cell, by an atomic absorption spectrometer. In both cells, it was easy to get an efficiency of 99.9'70 and the result suggested good accuracy would be obtained. Furthermore, it appears that the cell can be used for the flow rate of up to 6 ml/min with an efficiency of more than 99.5%. Of course, an agreement was obtained between observed and calculated values related to the flow rate and output current. Applications. The first application example is a detection of an alkaline earth group separated with cation ex-
* ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
, 0.4,!.~mol
Br, 0.6,umol
w
01
L
Y V
1 L
0
-
U
A
40 60 Time ( m i n )
20
Figure 7. Halide ions Resin: AG 1-X8, -400 mesh; column: 90 mm X 9 mm-i.d., room temperature; eluant: 2M NaN03-30% acetone (72); flow rate: 1 ml/min; working electrode: Ag; detection potential: 0.16 V vs. ferro-ferricyanide
change resin. The result is shown in Figure 5. The order of 10-7 mole can be detected with sufficiently high sensitivity. Heavy metal ions, Hg, Cu, Zn, Ni, Pb, Cd, and Co ions are also detected using the same electrochemical reaction (Figure 6). Figure 7 shows an example of the halide ions. The resin is anion exchange resin of -400 mesh and the eluant is 2M NaN03 of 30% by volume of acetone-water mixed solvent (12). The working electrode is silver gauze of 80 mesh. A chromatogram of amino acids is shown in Figure 8. Acetate and monochloroacetate buffer (13) were used as eluant. Carboxylic acids are also detectable with the secondary electrode reaction of p-benzoquinone and proton. Figure 9 shows that the acids were separated by H-form cation exchange column with methyl cellosolve-water as an eluant. Maleic, fumaric, formic, acetic propionic, n-butyric, isovaleric, n-valeric, isocaproic, and n-caproic acid were separated from each other. Phenols are detected by primary electrochemical oxidation. An example shown in Figure 10 presents a chromatogram of ortho-, meta-, and para-aminophenols, phenol, and cresols separated by cation exchange chromatography. Figure 11 shows an example of highly sensitive detection of phenols. In this case, the noise level is about 0.1 PA. Then, the detection limit of the coulometric detector is about 5 x 10-1' gram/sec for aminophenol. Figure 12 shows a chromatogram of sugars. The detection is based on an indirect constant potential coulometric technique-namely, sugars in the effluent are oxidized by basic ferricyanide a t 80 "C and consequently the ferrocyanide produced is detected by electrochemical oxidation. (12) G. Muto, Y . Takata and H. Tsuda, Nippon Kagaku Zasshi, 88, 432 (1 967). (13) M . C. Corfield and A. Robson. Biochem. J., 84, 146 (1962).
0
60 90 Time ( m i d
30
Figure 8. Detection of amino acids Sample size: 2.5 X 1 0 - 7 mole (hydroxyproline: 5.0 and alanine: 22.5 X mole); resin: Aminex A-4; column: 520 m m - X 9 mm-i.d., 40 "C; eluant: 0.125M CCIH2COOH-0.04M CH3COOH- 0.1M CH3COONa-0.1M NaCl (73);electrolyte: 0.01 M CuSCN-4M KSCN; detection pqtential: 0.39 V vs., Ag-AgSCN (2M KSCN)
lo-'
0
IO
20
30
40
50
60
Time ( min Figure 9. Chromatogram of carboxylic acids
-
Sample size: 0.5 2 X l o - ' mole; resin: Hitachi No. 2613; column: 530 mm X 9 mm-i.d., 54 OC; eluant: 10% methylcellosolve-water; flow rate: 1 ml/min; electrolyte: 0.01 M p-benzoquinone-0,001 M hydroquinone0.1M KN03; detection potential: 0.45 V vs. Ag-AgI
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
1867
m
Q)
OH
0
10
20
30
40 50 Time ( m i n 1
60
Figure 10. Phenols Sample size: 5 X l o - ' mole; resin: Hitachi No. 2613; column: 175 m m X 9 mm4.d.. 40 " C ; eluant: 0.2M K2HP04-20% EtOH, pH 9.5; flow rate: 1 ml/min; electrode: Pt; detection potential: 0.60 V vs. ferro-ferricyanide
t OH
A
I\ u 0 10 20 I
?
8 I
_-__
1
30
60
90 120 Time ( m i n 1
Figure 12. Monosaccharides
____
30 40 Time (rnin)
Figure 11. High sensitive detection of phenols Sample size: 5 X 1 0 - l o mole; resin: Hitachi No. 2613; column: 70 mm X 9 mm-i.d., room temperature; eluant: 0.4M K~HP04-20% EtOH, pH 8.8; flow rate: 1 ml/min; detection potential: 0.50 V vs. ferro-ferricyanide
CONCLUSIONS I t should be pointed out that, when constant potential coulometry is applied to liquid chromatography, the following advantages, in particular, are expected: 1) The sensitivity is high. 2) So far as the current efficiency is loo%, the preparation of a calibration graph is unnecessary. 3) Even such material as is not separated by liquid chromatography can be quantitatively analyzed by selecting the electrode potential. 4) By introducing secon-
1868
0
1
mole; resin: AG 1-X4, -400 mesh: column: Sample size: 2.5 X 115 m m X 9 mm-i.d., 65 "C;eluant: 0.6M H3B03-NaOH, pH 8.8; flow rate; 1 mljmin: electrolyte: 0 . l M K3Fe(CN)6-3M NaOH; reaction coil: 8 m X 0.8 mm-i.d.; reaction temperature: 80 "C; detection potential: 0.08 V vs. ferro-ferricyanide
dary electrochemical reactions, even electroinactive substances can be detected. 5 ) The variation in the flow rate and temperature of the effluent hardly affects the results of analysis.
ACKNOWLEDGMENT The authors wish to thank Y. Arikawa of Hitachi Research Laboratory for his kind advice, Y. Hamano of Naka Works, Hitachi, Ltd., for his technical assistance in instruments, and M. Taki, Miss M. Fujieda, and Miss T. Saji for their experimental assistance. Received for review October 11, 1972. Accepted February 20, 1973. Presented a t the IUPAC International Congress on Analytical Chemistry, Kyoto, Japan, April 1972.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
